The long-term objective of this work is to elucidate the biological mechanisms underlying the therapeutic effects of acupuncture, with focus on the local tissue effects of acupuncture needling. Acupuncture therapy involves manipulation of the subcutaneous tissue with fine needles inserted through the skin at specific points, termed acupuncture points. Recent evidence suggests that the connective tissue in the vicinity of an acupuncture point plays a critical role in the local tissue response to in vivo acupuncture, and that acupuncture needling initiates a mechanotransduction response by deforming the connective tissue and its resident fibroblasts. The goal of the proposed project is to develop an in vitro model of the connective tissue region surrounding the acupuncture point that can be used to investigate the fibroblast response to forces created by acupuncture needling. We will use connective tissue equivalents based on collagen and fibroblasts. In Specific Aim 1, we will determine the effects of connective tissue geometry on the mechanical response of the tissue equivalents to acupuncture needling. It has been proposed that the organization of connective tissue at perimuscular fascial planes coincident with acupuncture points enhances the mechanical response to needling. We will establish an in vitro assay that mimics the perimuscular connective tissue geometry. We will replicate the tissue planes by generating a three-dimensional, fibroblast- populated collagen gel around rigid partitions. We will develop a microrobotic needling instrument to enable precise, reproducible needling and force measurement while also visualizing tissue movement. We will compare the response in the partitioned gel to that in identical gels without partitions. In Specific Aim 2, we will evaluate the effects of tissue geometry on short- and long-term fibroblast organization and morphology using time-lapse videomicroscopy to monitor spatial and temporal changes in fibroblast distribution, shape, and orientation in both of the assays developed in Specific Aim 1. In Specific Aim 3, we will characterize key fibroblast phenotypic changes in response to acupuncture needling that are associated with mechanotransduction, including integrin expression, MAP kinase phosphorylation, and matrix turnover. An in vitro assay of acupuncture will enable further study of the biological mechanisms of acupuncture and may ultimately contribute to improved application of acupuncture therapy. [unreadable] [unreadable] [unreadable]